Direct capture using large bead chromatography media

ABSTRACT

Disclosed is a continuous process in which a subset of a number of mutually identical columns, are connected in series. The process liquid, e.g. crude cell culture harvest, is supplied to the most upstream column of the subset. It flows successively through the in series connected columns and leaves the subset through the most downstream and flows into the downstream collection vessel. As soon as the packed bed of the most upstream column is become saturated with product, this column is disconnected from the subset. It is removed from the series connection. A replacement, identical, column is added such that it is connected in series downstream from the most downstream column of the subset. This process is repeated.

This application is the U.S. national phase of International ApplicationNo. PCT/NL2019/050038 filed Jan. 19, 2019 which designated the U.S. andclaims priority to NL Patent Application No. 2020305 filed Jan. 22, 2018and NL Patent Application No. 2022427 filed Jan. 21, 2019, the entirecontents of each of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to direct or primary capture (e.g. captureof product from crude feed) of a product present in a, preferablyunclarified (in other words not cleaned), process liquid using a column(e.g. radial flow type) for liquid chromatography comprising a packedbed of large beads. In particular the invention relates to downstreamprocessing of biologics from cell culture or cell fermentation harvestsand to a related liquid chromatography system.

Description of the Related Art

It is often necessary or desirable to fractionate a fluid mixture toseparate out or isolate useful or desired components. This can beachieved by using liquid chromatography systems.

Chromatography systems of various sizes are used in both laboratoryanalysis operations and for industrial scale production operations inwhich separation steps such as fractionation from human blood orcapturing or removing impurities from a pharmaceutical can be carriedout on a large scale in a batch process and nowadays also in a continueprocess.

Liquid chromatography may briefly be described as the fractionation ofcomponents of a mixture based on differences in the physical or chemicalcharacteristics of the components. The various liquid chromatographicsystems fractionate the components with a fractionation or solid matrix.Some liquid chromatographic matrix systems fractionate the components ofa mixture based upon such physical parameters as molecular weight. Stillother liquid chromatographic systems will fractionate the components ofa mixture based upon such chemical criteria as ionic charge, hydrophobicnature, and the presence of certain chemical moieties such as antigenicdeterminants or lecithin-binding sites on the components, togethercharacterized as “affinity” moieties.

Liquid chromatography often uses a separation column. The separationcolumn contains a stationary phase, packing, packed bed or matrix mediumor material, as well known in the art, which interacts with the variouscomponents of the sample fluid to be separated. The composition of theseparating medium depends on the fluid being directed there through soas to produce the desired separation.

In order to prepare a chromatographic column to be able to separatebiological substances effectively, it is usual to pack fine particles ofseparation material as tightly and as uniformly as possible in thecolumn tube, the packed bed. Filling of the column, or column packing asit is often referred to, is normally effected by closing one end of thecolumn with an outlet means which includes a filter element, and pouringor pumping under pressure a liquid suspension of the particles into thecolumn from the other end of the column. Whereas the pumped liquid isable to pass through the filter element essentially unobstructed, theparticles are retained by the filter element, so as to buildup aparticle bed along the length of the tube. As the column tube is filled,the particles are pressed out towards the wall of the tube and theparticle bed obtains a stable compaction state with the particles welldistributed by the pressure generated by the pump or compression of thetop filter, this state being maintained during the whole of the fillingprocess.

The first type separation columns generally known in the art are of acylindrical construction and the fluid flows axially through aseparating medium bed (packing or matrix) retained in the column. Themedium bed is retained between supports or frits on either or both endsof the column. As the sample (also called “feed”) or elution (alsocalled “desorption”) fluids pass through the separating medium bed, theconstituents of the relevant fluid travel at different rates due totheir different interaction with the matrix or packing material. As aresult, these constituents emerge separated (i.e., have differentelution times) in the outlet stream of the column.

With the need for high performance, low pressure chromatography,horizontal or radial flow type chromatographic columns were developed.Such horizontal or radial flow columns are, e.g., described and claimedin U.S. Pat. Nos. 4,627,918 and 4,676,898. In the horizontal or radialflow type columns, the sample/adsorption and elution/desorption fluidsare introduced via a distributor to the outer periphery orcircumferential wall or surface of the separating medium or matrix,which consists of packing material, where the components get separatedand the fluids pass horizontally or radially inwardly through theseparation medium to a central or collection port and then elute fromthe column at different times and at different rates. This horizontalflow column design has a high cross sectional area and very loweffective bed height. It thus offers the ability to handle very highflow rates at low operative pressures.

As used herein, the terms “horizontal or radial flow mode”, which areused interchangeably, are defined as flow of the sample (e.g.biomolecules) or eluant or wash fluid through the chromatographic columnin a direction that is perpendicular to the longitudinal axis of thecolumn, regardless of the position of the column relative to the workbench or support stands or other equipment used to support or stack thecolumns.

This chromatographic separation in a horizontal mode may be accomplishedby means of a chromatographic column constructed so as to have an innerand outer annulus, with the matrix material being packed there between,thus having a torus or doughnut shape. The bed height is thus computedas the distance between the inner and outer annuli. Chromatographyconsequently takes place radially in the column.

This horizontal mode column configuration results in even bed heightsince the inlet and outlet distributors are fixed. The distributor andcollection channels are designed to provide even application of thesample and horizontal streamlines across the chromatographic bed. Thelong, vertical column assembly with horizontal flow is easy to fabricateand convenient for packing and handling. Furthermore, since the bedheight is constant along the length of the column, both thecross-sectional area and bed volume are proportional to the columnlength. Thus, scale-up is possible by linearly increasing the length ofthe column in proportion to the desired scale of operation. At any scaleof operation, the pressure drop remains constant and scale up isaccomplished easily by linear increments of the column bed length.

The horizontal mode column is particularly applicable to highperformance low pressure chromatography used in conjunction with theseparation of biomolecules like proteins or other organic or inorganiccompounds particularly sensitive to shear forces. This column type iscapable of use both in high performance and conventional chromatography,in the identification and separation of mixtures in the preparativemode, and also provides scale-up to larger columns for use in theisolation of components from mixtures in quantities suitable forindustrial or production purposes.

The development of chromatography columns has aimed at providing ease ofoperation and various additional benefits which have particularcommercial importance. These include: (a) the ability to be sterilizedby autoclaving; (b) improved sanitation by virtue of design featuresgiving less carryover of product from one batch to the next; (c) theability to resist solvents; (d) material conformity to food grade FDAregulations; (e) an improved pressure tolerance; (f) lower cost; (g) thepotential for full or partial automation; (h) the ability to providedisposable columns; (i) linear process upscale.

During use, the performance of the packed bed, i.e. the beads, isconstantly deteriorating. Since the beads typically are extremelyexpensive (in the order of eurol-20/millilitre), at least for industrialapplication, the packed bed should be used-up completely. The samepacked bed is preferably used many times (200 times being the practicallimit) and cleaned thoroughly before each next time (i.e. between eachtwo successive uses). Despite all care, the theoretical maximum numberof uses is hardly matched in case of use that covers a period of manymonths (say 6 or 12 months), due to aging. Aging is aggravated due tothe inevitable inability of complete removal of any possiblecontamination during cleaning between two subsequent uses.

BACKGROUND SPECIFIC TO DIRECT CAPTURE

Now, information more specific to direct capture is provided by way ofmammalian cell culture as an illustrative example of biomolecules.

Bio-pharmaceutical products, often therapeutic proteins can be producedby a multitude of organisms. The most common production method today ismammalian cell-culture in which the micro-organisms are grown and areprogrammed to produce an extra special compound that normally would notbe produced by this (e.g. CHO) cell. The therapeutic product programmedfor is the target Active Pharmaceutical Ingredient (also called API).Since many biological compounds carry the signature of the (non-human)host, a product produced by a non-human cell can cause rejection by thereceiver, cell-culture cells are there for “humanized”. This is done byfusion with mammalian cells to yield the human signature and is calledmammalian cell culture. The mammalian cells are grown in a big containerin which a carefully balanced power-cocktail, the culture media,supports their growth. The media contains all nutrients to getexponential cell growth. When the cell is programmed well, it willproduce the compound of interest in large quantity and when possible,excretion of the API into the surrounding media (extra cellular). Atypical cell culture volume for API production is 1,000-10,000 litre.

A drawback of the these perfect miniature machines is the fact that thefusion cells are more vulnerable. The cells must be treated gently toprevent breaking (in other words they have to be kept viable). Brokencells result in two issues: the cells stop producing, but more importanttheir internal mass is excreted into the culture media. The Host CellProtein (HCP) and Host Cell DNA are contaminating the media which makespurification much more difficult and digestive enzymes from the internalcell are starting to digest all protein in the media, i.e. also thetarget API. During cell culturing mixing is done gently and evensparging with air can cause cell breakage. When the cell culture reachesits limit in growth, the culture is harvested. Harvesting the1,000-10,000 litre cell culture in short time without rupturing thecells is the big challenge.

Current state of the art at industrial scale is rapid clarification,centrifugation and/or filtration of the cells, isolating the cells-massfrom the surrounding media (the plasma) that is containing the API.Unfortunately also centrifugation and filtration cause cells to ruptureand thus contamination of the plasma. Unclarified/crude e.g. meanswithout filtration and/or centrifugation of the to the column fedprocess liquid.

A few decades ago Expanded Bed Adsorption (EBA) was invented to offerprocessing of the crude feed without clarification. This process issometimes called “fluidized bed adsorption”. Through the bottom of asettled slurry of chromatographic beads, the cell culture is fed intothe bed. There is no limiting sieve or other restriction above thesettled beads and the bed is allowed to expand upward by force of theupward flow. The columns are tall and to allow the bed to expand freely,when settled, only about ⅓ is filled with the settled beads. The absenceof resistance however caused preferential paths and therewith patches ofstagnant accumulating cells causing contamination outside the paths. Toprevent this, the chromatographic beads are artificially made heavier byincluding a metal core. So the settled beads are heavy creating adensity (resistance) i.e. a dynamic pore-size reduction, forcing thecells to evenly distribute over the entire surface of the bottom andforcing the beads to make room for passage. Although this techniqueworks well in some cases, it is difficult to control and often is notvery robust.

PERTINENT BACKGROUND ART

U.S. Pat. No. 5,466,377A (Grandics et al., published 1995) discloses theuse of large bead chromatography particles in standard or conventional,low pressure, packed bed chromatography columns for the direct captureof product from an unclarified process liquid as a system for downstreamprocessing of biologics from a different type of homogenized cellculture, microbial cell culture or bacterial cell fermentation harveststhat employs micro-organisms that are about 0.5 to 1 micrometre and atleast 10 times smaller than mammalian (CHO) cells. Disclosed is the useof an axial type column of 1.5 centimetre diameter with end-platescreens with large pores (60-180 micrometre, equals 0.06-0.18millimetre, pores), beads having a diameter of 100-300 or 300-500 or500-800 or 800-11000 micrometre (equals 0.1-0.3 or 0.3-0.5 or 0.5-0.8 or0.8-1.1 millimetre, respectively), and a bed volume of 9 millilitre. Nofurther information is provided for the column. However from the dataprovided for column diameter and volume the “bed height”, meaning theaxial distance between the end-plate screens, can be calculated andequals 5.0 centimetre. At this small scale of operation in anexperimental laboratory environment, the in here mentioned problems,e.g. relating to contamination, aging and completely using-up of thepacked bed for economy reasons, generally are absent. However, attemptsfor full scale production at economic level showed that the axial columntype is unsuited for industrial application of direct capture even withthe demonstrated homogenates and much smaller bacterial cells.

SUMMARY OF THE INVENTION

The object of the invention is versatile and, according to a firstaspect, to provide an improved, contamination free, low pressure, gentleacting packed bed chromatography column packed with large beads foreffective direct capture of product, e.g. biomolecules) from anunclarified (or crude) process liquid, and related process, inparticular for downstream processing of biologics from mammalian CHO orsimilar cell culture or cell fermentation harvests (cell culture densitytypically above 0.1 or 0.5 or 1 or 2 and/or below 50 or 100 millioncells per millilitre). In a second aspect the first downstreamprocessing step, the primary capture of said high density cell cultureharvest, is in the radial flow packed bed capture column, directly fromthe cell culture vessel without any intermediate, filtration or otherclarification equipment, nor any harvest holding-step or holding vessel.The processed cell culture liquid, depleted from the product ofinterest, is leaving the primary capture column (typically containingcellular/particulate content) directly into a, e.g. waste or collectionvessel. By the removal of the filtration equipment, a reduction ofcapital cost and setup time is achieved, while the removal of theharvest holding vessel reduces the available incubation time forproteolytic attack and autodigestion of the product of interest by hostcell enzymatic activity during its residence time in the vessel andhence removing the vessel will reduce the risk of product damage ordigestion. Avoiding cell damage is also an object. Further objects canbe found elsewhere in this disclosure.

According to the invention the solution is provided by application of ahigh performance chromatography column that is characterized by a packedbed and by horizontal or radial flow through the packed bed. In here,this direct capture technology using radial flow is also called cTRAC(cell Tolerant Radial Affinity Chromatography). It will be appreciatedthat a column designed for radial flow typically means that the packedbed is torus or doughnut shaped, although alternative designs are alsocovered, preferably designs which mimic the flow conditions of a radialflow type column, e.g. wedge or conical shaped, wherein the surface area(I) of the one frit is bigger compared to the other (O), to suffice thefunctional I/O requirement. Typically, the frit through which the liquidto be processed enters the packed bed, also called the inlet frit, hasthe larger surface area compared to the frit through which the liquidexits the packed bed, also called the outlet frit. Thus, the liquid tobe processed typically flows radially inward from the inlet frit towardsthe outlet frit. The typical upstream holding vessel can also be avoidedsuch that the liquid from the cell culture can be directly fed to thechromatography column.

Surprisingly, the present inventor discovered that a properly designed,properly balanced column of radial type can create the additionaldynamic resistance required to result in even distribution of the liquidover the entire surface area without reduction of pore-size. The cTRACradial column features an inlet surface area which is larger than theexit surface area. The packed bed is radial-shaped, wedge-shaped orconical shaped. The packing density of the packed bed is properlycontrolled by the annular packing method resulting in an evendistribution of bead-porosity all over the packed bed. The interstitialvolume between the packed beads is identical at the inlet (the largersurface) as compared to the outlet (the smaller surface). Therefore theinterstitial pore-size is constant across the entire column. Also, thereis no need for a clarification and/or harvest holding step prior to thecolumn and during flow of the process liquid through the column, theliquid leaving the column (typically cellular/particulate content) canbe fed directly into a, e.g. waste, collection vessel. If processing ane.g. high density continuous-perfusion, cell culture, in which theculture cells are e.g. retained within the cell culture upstream thecolumn, resulting in a cell culture with extreme cell densities and inwhich only a relative small volume containing a limited amount of cellmatter continuously is send to be processed downstream, the column canbe fed, e.g. connected, directly by/to the cell culture exit with theoption to either recycle the feed via the effluent (partly) back intothe upstream source, e.g. continuous-perfusion cell culture, or send thecolumn output into the, e.g. waste, collection vessel.

This solution was a surprise since passing cells (typically 5-20micrometer) through a packed bed without the problems mentioned above(e.g. blocking, contamination, economy of use) was always consideredimpossible for industrial or rational application. On the one hand thebigger beads packed into a homogenous matrix, leave an “open space”between them, the interstitial volume, which is beneficial. Through thisinterstitial volume the cells are able to flow with only limited or evenwithout obstruction (resistance, shear forces). The low or even lackingresistance is good for cell viability, cells are flowing gently with lowor even without obstruction between the beads towards the exit of thecolumn. The driving force for cell travel is the flow of liquid.However, the low or even lacking resistance also means a tendency forpreferential paths, cell accumulation and contaminating patches andcomplete blocking like in the earlier EBA example.

Likewise and in parallel to the earlier EBA example, the main challengein the application of a packed bed chromatography is to force theincoming liquid to evenly distribute over the entire surface preventingpreferential paths, without creating pore size reduction since in apacked bed this would be static and would result in immediate columnblocking. In the case of earlier reported attempts with traditional(axial) chromatography columns with packed bed, columns would eitherblock and/or contaminate.

Without being bound to theory, it is believed that the surprising effectof the present invention can be explained by the equation ofKozeny-Karman (viz., e.g., Besselink: Are axial and radial flowchromatography different?; Journal of Chromatography A; 1271(2013)105-114). According to this equation, the increase in the superficialvelocity causes an increase in resistance to flow between the beads.Further, the increase in superficial velocity seems proportional to theI/O-ratio (sometimes referred to as “α” or “alpha”) which is directlyrelated to the shape of the radial column.

The decrease of surface area towards the exit of a radial flow typecolumn results in a local flow velocity increase, which withoutreduction of the interstitial space causes an increase in resistancewhich in turn causes an even distribution of liquid over the wholesurface. Although the surface at the exit is smaller, focusing theliquid flow like an optical lens, the even distribution is transferredto the larger surface at the entry. In stead of a radial inward flow, aradial outward flow through the packed bed could be applied for theinventive column.

Also another issue that is absent in case of the EBA but indeed presentin case of packed bed columns is the sieve (frit) in between which thebed is packed and that separates the internal volume from the outsideflow-path. Experiments have shown that only a very thin and open/smoothstructured frit will allow cells to pass without rupturing or differentdamage.

The thin and open structured frits of the preferred radial flow columnallow superior cell viability. The cylindrical form of the radial columnallows the frits to be thin since the shape stability of a cylindricalobject is many times that of a flat surfaced object with the samematerial thickness. Thinner frit causes more viability and less rupturedcells.

The bead size, bead rigidity, packing density, inlet-surfacearea/outlet-surface area ratio (I/O-ratio) and the volumetric flow rate,now are some of the possible parameters for the proper dynamicresistance to support even distribution of the liquid to be processed.The proper balance between these parameters have to be optimized foreach individual application. Then the resulting dynamic resistance tosupport even distribution is determent for the application.

Preferably, for the frits one or more of the following applies: madefrom stainless steel; electro polished surface; hydrophilic surface;comprising at least or exactly one or two or three or four layers orsheets of woven wires of stainless steel, directly laid on top of eachother, at least one of the sheets, e.g. the filter layer, preferablywoven according to a plain weave or twilled weave or plain dutch weaveor twilled dutch weave or reversed plain dutch weave or reversed twilleddutch weave or five-heddle weave pattern; the sheets provide a unitedassembly, preferably are mutually sintered (also called diffusionbonded); at least one or two, e.g. each, sheet is woven from wireshaving a diameter at least 10% or 20% different, e.g. larger or smaller,from the immediately adjacent sheet; the wire thickness among sheetsincreases from the one to the other face of the frit, preferably fromthe face facing and/or delimiting the packed bed; the wire thickness ofthe sheets is at least 25 or 50 micrometre (equals 0.025 and 0.05millimetre) and/or not more than 500 micrometre (equals 0.5 millimetre);at least one or two, e.g. each, sheet has a pore size at least 10% or20% different, e.g. larger or smaller, from the immediately adjacentsheet; the pore size among sheets increases from the one to the otherface of the frit, preferably from the face facing and/or delimiting thepacked bed; the sheet, e.g. reinforcement sheet, having the thickestwires and/or largest pore size, e.g. at least 500 micrometre (0.5millimetre) is the, preferably ultimate, inner- or outermost sheet ofthe frit, preferably the sheet the most remote from the packed bed;thickness at least 0.3 or 0.8 or 1.0 millimetre and/or not more than 1.2or 1.5 millimetre; pore size (this is the “nominal” pore size, definedby the diameter of the largest rigid sphere that can pass the pore) atleast 50 or 100 and/or not more than 200 or 500 micrometre (equals 0.05,0.1, 0.2 and 0.5, respectively, millimetre); welded, preferably by acontinuous weld bead, to the axial end plate; the number of sheets ofthe inner frit is one or two or three more compared to the outer frit;contains a single filter layer; a filter layer is directly exposed tothe packed bed; at the side of the filter layer facing the packed bed, alayer, e.g. protective layer, is absent; a filter layer provides thesurface layer; has at the one side of a filter layer no layer or merelya protective layer and at the other side merely a protective ordispersion layer and possibly exactly one or two further layers,preferably reinforcing layers.

Preferably, the I/O ratio is at least 1.5:1 or 2:1 or 2.5:1 and/or notmore than 3:1 or 3.5:1 or 4:1 or 5:1 or 10:1. I/O is the ratio betweenthe radius (illustrated in FIG. 1 by arrows R1 and R2) of the outer andinner frit of the column.

Bed height preferably is at least 10 or 20 and/or not more than 100 or150 or 200 or 300 millimetre. Bed height (illustrated in FIG. 1 by arrowH) is the distance between the inner and outer frit or, in differentwords, the difference between the diameter of the inner and outer frit.Bed volume preferably is at least 10 or 25 or 50 or 100 or 200 or 250 or500 millilitre and/or not more than 10 or 20 or 150 or 250 litre.

The diameter (in millimetre) of the inner frit preferably is: at least10 or 15 or 20 and/or not more than 80 or 100 or 150 or 300, e.g. 23 or50 or 60 or 100.

Alternatively, in stead of a complete doughnut or torus shaped bed tocontain the beads, a segment of it is used (viz. FIG. 13).

In the typically cylindrical space delimited by the inner frit, a coremember is preferably located, the external wall of which delimits aninner flow channel with the inner frit.

Preferably the outer flow channel delimited outside the outer frit has awidth at least 0.5 millimetre and/or not more than 2 or 3 or 5 or 10millimetre. Preferably the inner flow channel has a width at least 0.5millimetre and/or not more than 2 or 3 or 5 or 10 millimetre, morepreferably equals the outer flow channel width times the I/O ratio (alsonamed “I/O-width”) and/or its width is at least 0.5 or 1 or 2 and/or notmore than 5 or 10 millimetre wider than the outer flow channel width. Incase of a conical core member the inner flow channel width preferably isat least 0.5 and/or not more than 1 or 2 millimetre at the one axial endand at least 1 or near I/O-width and/or not more than 3 or 4 or 6millimetre at the opposite longitudinal end (“longitudinal” is parallelto the axial direction of the column) and/or is at least 1 or 2 and/ornot more than 3 or 5 or 10 millimetre wider than the narrow axial end.

Preferably, the invention is applied in a continuous process in which asubset of two, three or more, e.g. five, columns, preferably mutuallyidentical, are connected in series, preferably not more than 5 or 10.For the process, the number of columns required for the complete setequals the number of columns of the subset, plus one, two, three or morecolumns, preferably identical to the columns of the subset. The processliquid, e.g. crude cell culture harvest, is supplied to the mostupstream column of the subset, flows successively through the in seriesconnected columns and, e.g. as from the target product depleted cultureand cells, leaves the subset through the most downstream and flows e.g.into the downstream, e.g. waste, collection vessel. As soon assufficient time or a predetermined quantity of time has elapsed, e.g.the time at which the packed bed of the most upstream column hascaptured a predetermined quantity of product, e.g. is become saturatedwith product, the most upstream column is disconnected from the subsetand removed from the series connection and a replacement, identical,column, preferably containing a fresh or reset packed bed, is added suchthat it is connected in series downstream from the most downstreamcolumn of the subset and this process is repeated. During the timerequired to saturate the most upstream column of the subset with targetfrom the process liquid, the most recently from the subset disconnected,saturated column is off-line processed to reset the packed bed (e.g.wash, elute/desorb, clear and prepare the bed) to become available foraddition to the subset to become saturated again. The column with lowresistance to flow allows this off-line process to be performed at muchhigher velocities, possibly up to 10 times the flow rate through thesubset. This creates a time-lapse, responsible for a reduction of theminimum number of columns required and therewith reduces the totalvolume of the complete set. This cycle is repeated several times suchthat repeatedly after time elapse a most upstream column is removed fromand a most downstream column is added to the in series connected subsetof columns such that the number of columns of the subset remains thesame during the complete process. Typically, the column removed from theseries connection has its packed bed processed, e.g. cleaned, to becomereset and thus fit for returning to the series connection, initially asthe most downstream column and subsequently obtaining successively theupstream rankings as other reset columns are added downstream. The resetprocedure is preferably started and completed within the time elapsebetween removing two successive columns from the series connection, suchthat a column is only as briefly as possible removed from the seriesconnection and the least possible number of columns is needed.Preferably, the number of columns being part of the continuous processis the number of columns connected in series (being the subset) plus asingle column in the reset procedure plus possibly one or two sparecolumns.

The columns of the subset are preferably mutually connected such thatthe process liquid flows through the packed bed in the same directionfor all columns (i.e. radially inward or outward), however in analternative at least one of the columns has its packed bed flown throughin the direction opposite to at least one or two other columns of thesubset. E.g. of the subset the most upstream column has its packed bedflown through in radially inward direction and a more downstream columnof the same subset has its packed bed flown through in radially outwarddirection.

For the same volumetric yield of captured product, the continuousprocess requires less volume of beads, e.g. at least 25% or 50% or 75%less, compared to the batch process.

The packed bed volume of each column associated with this continuousprocess, is preferably at least 25 or 100 or 250 millilitre and/or notmore than 10 or 20 or 100 litre.

An object of this continuous process is the significant reduction ofecological footprint, setup time, process time, waste materials, timeand space spend in the clean room.

The column, preferably the subset in the continuous process, could beused to remove an, e.g. growth rate limiting, contaminant from theprocess liquid, wherein the contaminant is the target for the packed bedand the liquid leaving the column or subset is e.g. as culture mediasupplied to a cell culture.

With a continuous process, it is very important that the process liquidthat leaves a more upstream column, is still free from contaminations,e.g. damaged cells or cell contents such as protein or DNA. This can beprovided by designing the outlet frit such that cells can pass it damagefree. For both the continuous and batch process, the inlet and/or outletfrit is preferably designed such that cells can pass it damage free.

The beads preferably have a diameter at least 200 and/or not more than500 or 1000 micrometer (equals 0.2 and 1.0 millimetre) and/or arehydrophilic.

In an embodiment, the invention relates to a liquid chromatographycolumn, utilizing horizontal or radial flow of sample material passingthere through comprising one or more of: a housing defining a chambertherein and including at least one, preferably removable,axial/longitudinal end section; a first (outer) and second (inner)axially/ longitudinally extending porous frits or membranes positionedwithin said chamber of said housing; a bed or packing of, preferablyparticulate, chromatographic separation material positioned within saidchamber of said housing and intermediate said porous frits, the first orouter of said porous frits being adjacent a housing wall and definingwith said wall a cylindrical shaped ring type outer flow channel, thesecond or inner of said porous frits being positioned adjacent anoptional core member and defining with said core member a cylindricalshaped ring type inner flow channel; distribution means operativelyconnected to said outer flow channel; collector means operativelyconnected to said inner flow channel; a supply channel (also namedliquid inlet) operatively connected to the distribution means and anexhaust channel (also named liquid outlet), operatively connected to thecollector means; said distribution means and said outer flow channelbeing constructed to direct associated material to be separated in saidbed evenly across an axial/longitudinal length of said bed in asubstantially horizontal direction.

Further, said porous frits are coaxially positioned with respect to oneanother. Practically, said first porous frit having a largercross-section than said second porous frit and said core member, ifapplied, is centrally located in said housing chamber coaxial with thefirst and second frit.

Preferably, one or more of the following further features apply to theinventive column: one of the axial end sections, is penetrated by boththe supply channel (or liquid inlet) and the exhaust channel (or liquidoutlet); the exhaust channel is coaxial with the first and/or secondfrit; the outer flow channel is radially spaced from the housing centreand/or the exhaust channel; the housing has a substantially cylindricalwall; the first and second frit are part of a cartridge removablycontained in the housing; at the one axial end the first and second fritare connected by an end wall closing the space between the first andsecond frit at said axial end and possibly having a passage for theexhaust channel or core member, and/or at the opposite axial end theinner flow channel is closed by an end wall connected to the secondfrit; the outer and/or inner flow channel extends substantially thecomplete height of the chamber; the packed bed, in the operativeposition within the chamber, extends from the end section opposite theaxial end wall beyond the axially extending housing wall; an outflowchannel, connecting to one or more of the inner flow channel, thecollector space, the exhaust channel, extends inside, preferablylengthwise of, the core member and/or debouches at theaxial/longitudinal end of the core member within the chamber; the coremember extends substantially the complete height of the chamber; thecore member penetrates an axial end section; the core member projectsfrom the axial end section; the axial end section has a central borepenetrated by the core member; the core member keeps with itsaxial/longitudinal free end a gap with the associated axial end sectionand/or tapers to a narrow dimension towards said axial end section; thedistribution means comprise a radially outward narrowing, preferably thecore member circumventing distribution space, preferably between theaxial end section, and the space containing the packing and the outerflow channel preferably connects to, more preferably debouches into,said distribution space; the cross sectional area of the inner flowchannel increases, preferably continuously, along the axial direction,preferably due to the taper of the core member; one of the axial endsections, preferably the one associated with the axial free end of thecore member, contains a means, such as a closable fill port, to supplythe space between the frits with packing material for column packingpurposes, while the column is completely assembled (e.g. a fill systemaccording to WO2007136247 (Raedts); between the core member and at leastone of the axial end section and the cartridge or the end wallconnecting the first and second frit a seal is present; the column has ahandy outer dimension in the order of one or five or ten litre contents,possibly even up to one hundred litre.

Preferably, the invention is directed to one or more of: a homogenouspacked bed matrix of adsorptive beads with defined bead size, to createa controlled-porosity matrix over the entire packed bed such that itenables unhindered passage of concentrated unclarified mammalian (or anyother) cell culture harvest; cell culture harvest cell-diameter isapproximately 0, 1-10% of bead diameter; mean bead diameter isapproximately 120-500 μm; CHO cells are typically about 5-15 or 20 μm;homogenous distribution of cell culture cells within the porous matrixis resulting from the flow focusing brought about by the increase inresistance as explained by the Kozeny Carman equation as expressed bythe radially packed bed; the velocity increase travelling from inlet tooutlet, or decrease when travelling in reverse direction, of the radialpacked bed column creates the resistance that drives thefocusing/homogenous distribution of the cells without reducing theporosity between the bead matrix within the packed bed; a multi column(semi) continuous operation, in which a subset of serially connectedcolumns continuously is being loaded with cell culture harvest.

When operating and processing an e.g. high density“continuous-perfusion” cell culture, the majority of culture cells areretained within the cell culture upstream the column, resulting in acell culture with extreme cell densities and in which only a relativelysmall volume containing a limited amount of cell matter continuously issend to be processed downstream. The column can be fed, e.g. connected,directly to the cell culture exit with the option to either perform theprimary capture of product while sending the column efluent to thecollection vessle or recycling the feed via the effluent (partly) backinto the upstream source, alternatively the column can be packed with abig bead adsorbent programmed to capture/remove a e.g. growth ratelimiting, contaminant from the continuous perfusion cell culture whilerecycling cleaned media back into the cell culture.

When used for primary capture the column is exchanged for a clean one toallow recovery of the product. Alternatively, the saturated columncapturing the contaminant can be exchanged for a clean one to allow thecontaminated column to be cleaned. Thus also this process is designed asa continuous process.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated and form a part of thespecification, illustrate an embodiment of the invention and, togetherwith the description, serve to explain the principles of the invention.Shown is in:

FIG. 1-3 examples of the column in sectional side view;

FIG. 4 the column of FIG. 3 in top view;

FIG. 5 another column in perspective view from below, partly cut away;

FIGS. 6 and 13 two examples of a continuous process wherein a subset ofthree columns in series are applied;

FIGS. 7(a-d) and 8(a-c) illustrate weave patterns for a layer of a frit;

FIG. 9 a perspective, exploded view, of a prior art frit;

FIG. 10-11 sectional side views of prior art frits;

FIG. 12 a perspective view of a torus shaped filter bed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following reference numbers are used: column 1; cylindrical housingwall 2; axial housing end plate 3; seal 4; liquid inlet 5; liquid outlet6; packed bed 7; inner flow channel 8; packed bed fill opening 9;connector 10; fill tube 11 for packed bed; seal 12; seal 13; outer flowchannel 14; core 15; inner frit 16; outer frit 17; axial bed end plate18; distribution space 19; collector space 20; outflow channel 21;liquid outlet 22; bead height H; outer frit radius R1; inner frit radiusR2; axial direction arrow A (FIG. 3). The radial direction isperpendicular to the axial direction.

Each of the liquid chromatography columns shown in FIG. 1-5 comprises: ahousing, of cylindrical shape, defining a chamber therein and includinga removable axial end plate 3 of circular shape; a first (outer) andsecond (inner) porous frits 16, 17 or membranes of cylindrical shape; abed 7 or packing of particulate chromatographic separation materialpositioned intermediate said porous frits; optionally an axiallyextending core 15. The axially extending cylindrical external housingwall 2, first 17 and second 16 frit and core 15 are coaxial.

The first or outer frit 17 is adjacent the axially extending cylindricalexternal housing wall 2 and defines with said wall a cylindrical shapedring type outer flow channel 14, e.g. 0.5 millimetre wide. The second orinner frit 16 is adjacent the core member 15 and defines with said coremember a cylindrical or conical shaped ring type inner flow channel 8,e.g. 1.0 millimetre wide. In FIG. 1 a core member 15 is absent.

The axially top end plate 3 is, in an embodiment, penetrated by both asupply and exhaust channel 5, 6. These channels are coaxial with thefrits.

The first and second frit can be part of a cartridge removably containedin the housing.

At both axial ends the first and second frit are connected by a radialextending end wall 18 closing the space between the first and secondfrit 16, 17 to capture the doughnut shaped packing providing the filterbed.

The filter bed 7 plus the core 15 almost completely fill the housing 1.Between the top face 18 of the filter bed 7 and the bottom face of theaxial end plate 3 there is a distribution space 19 into which the outerflow channel 14 debouches. This distribution space possibly tapers inthe radial outward direction and merges at the thus narrowed radialouter circumference with the circumferential extending outer flowchannel 14. The circumferential inner flow channel 8, surrounding thecore 15, tapers in the axial direction from the one to the oppositeaxial bed end plate 18 along the core 15 and merges at the lower end ofthe core with a collector space 20 delimited between the lower core endand the bottom end 3 of the housing or the end wall 18. This taperedshape of the inner flow channel 8, providing a wide and a narrow axialend, is due to the axial taper of the core. The taper of thedistribution space 19 and of the inner flow channel 8 optimises flowcharacteristics. An outflow channel 21 extends lengthwise through thecore 15 (FIG. 2) and connects to the liquid outlet 6 and the collectorspace 20.

FIG. 3 shows an embodiment wherein part of the liquid entering throughliquid inlet 5 exits the column 1 without passing the outer frit 17, butin stead flows via the outer flow channel 14 to the liquid outlet 22,thus exits the column 1 without being processed. In an alternativeembodiment such liquid outlet 22 could be absent and the outer flowchannel 14 could be sealed at its axial end remote from space 19, e.g.by connecting the wall 18 to the wall 22.

The bottom end of the filter bed 7 contains a centrally located closablefill port 9, to supply the space between the frits 16, 17 with packingmaterial for column packing purposes. This fill port 9 and associatedseals and parts 10 and 11 could be absent, e.g. if the filter bed 7 isfilled with packing material in a different manner.

O-ring type seals are applied to seal the core to the axial end plateand the filter bed and to seal the lid to the axial extending housingwall.

FIG. 7a shows plain weave (PL), FIG. 7b twilled weave (TL), FIG. 7cplain dutch weave (PDW), FIG. 7d twilled dutch weave (TDW). FIG. 8ashows reversed plain dutch weave (PZ), FIG. 8b reversed twilled dutchweave (KPZ), FIG. 8c five-heddle weave (FHD). FIG. 9 shows a four layer(from top to bottom: protective layer, filter layer, distribution layer,single reinforcement layer), FIG. 10 a five layer (double reinforcementlayer) and FIG. 11 a six layer (triple reinforcement layer) frit.Compared to the frits of FIG. 9-11, a frit of the invention is obtainedas follows: In case of FIG. 9, by cancelling one or both of the top andbottom most layer (as seen in the drawing). In case of FIG. 10 or FIG.11 by cancelling at least one of the top layer (protective layer) andone, two or all of the reinforcing layers. FIG. 12 illustrates a bedsegment taken from the torus shaped bed filter.

The column operates as follows: Fluid is introduced through the supplychannel into the distribution space and from there flows radiallyoutward towards the inlet channel. In the inlet channel the fluid flowsaxially downward to be evenly distributed across the complete surface ofthe outer frit. Then, passing the outer frit, the fluid flows radiallyinward through the packing to arrive at the inner frit. Subsequently thefluid flows evenly distributed across the complete surface of the innerfrit through the inner frit to arrive into the outlet channel. The fluidflows axially downward through the outlet channel, along the outer faceof the core to be collected in the collection space. From there thefluid flows into the exhaust channel. If the core contains the exhaustchannel, e.g. as in FIG. 5, the fluid flows axially upward through thecore.

Further embodiments are also covered by the attached claims. E.g. theflow direction of the introduced fluid can be opposite, for which thesupply, exhaust, inlet and outlet elements are interchanged. Alsodifferent embodiments belong to the invention. Features of different inhere disclosed embodiments can in different manners be combined anddifferent aspects of some features are regarded mutually exchangeable.All described or in the drawing disclosed features provide as such or inarbitrary combination the subject matter of the invention, alsoindependent from their arrangement in the claims or their referral. Thedrawing, the specification and claims contain many features incombination. The skilled person will consider these also individuallyand combine them to further embodiments.

Conclusion: preferably a liquid chromatography column, utilizinghorizontal or radial flow of sample material passing there through,preferably in inward direction, comprising: a housing defining a chambertherein; a first and second axially or longitudinally extending porousfrits positioned within said chamber of said housing; a bed or packingof, preferably particulate, chromatographic separation materialpositioned within said chamber of said housing and intermediate saidporous frits, the first of said porous frits being adjacent said housingand an outer flow channel, the second of said porous frits beingpositioned adjacent an optional a core member and an inner flow channel;the bed is torus shaped; distribution means operatively connected tosaid outer flow channel; collector means operatively connected to saidinner flow channel, said distribution means and said outer flow channelbeing constructed to direct associated material to be separated in saidbed evenly across a longitudinal length of said bed in a substantiallyhorizontal direction preferably said porous frits are coaxiallypositioned with respect to one another, said first porous frit having alarger cross-section than said second porous frit, and said core memberis centrally located in said housing chamber.

1-15. (canceled)
 16. Method of processing a process liquid using a setof mutually identical liquid chromatography columns, each column is forliquid chromatography comprising a torus shaped packed bed of beads, theprocess liquid containing biologics that are captured by the beadswherein as the first processing step downstream from the process liquidcreating source, being a cell culture vessel, the process liquid is fedto a subset of at least two of the set of identical columns directlyfrom the source without any intermediate filtration or otherclarification equipment, nor any harvest holding-step or holding vesseland the process liquid, depleted by the subset of columns from theproduct of interest, is leaving said subset of columns after havingflown radially through each of the columns of the subset of columns,wherein the process liquid is concentrated crude CHO cell cultureharvest, the CHO cell diameter is approximately 0,1-10% of beaddiameter, the bead diameter is approximately 120-500 micrometer, thebeads are hydrophilic; each liquid chromatography column of the set isdesigned to be radially flown through by the process liquid andcomprising the packed bed of beads designed to capture product from theprocess liquid, the packed bed held between an inlet and outlet frit ofthe column wherein the inlet frit through which the liquid to beprocessed enters the packed bed, has a first surface area and whereinthe outlet frit through which the liquid exits the packed bed has asecond surface area smaller than the first surface area, wherein thecolumn has an I/O ratio which is the ratio between the surface area ofthe inlet frit and the surface area of the outlet frit, and wherein theliquid to be processed flows radially inward from the inlet frit towardsthe outlet frit; the columns of the subset of at least two columns areconnected in series with each other and the process liquid is suppliedto the most upstream column of the subset, flows successively throughthe in series connected columns and leaves the subset through the mostdownstream column and after time elapse, if the most upstream column isbecome saturated with product, the most upstream column is disconnectedfrom the subset and removed from the series connection and a replacementcolumn from the set is added to the subset such that a is connected inseries downstream from the most downstream column of the subset and thisprocess is repeated and the column removed from the subset is off-lineprocessed to reset the packed bed of this column such that this columnis made ready to become available for the subset to become saturatedagain by connecting this column in series as the most downstream columnof the subset.
 17. The method according to claim 16, wherein for eachcolumn the following applies: the I/O ratio is at least 1.5:1, and notmore than 4:1, such that the first surface area is at least 1.5 and notmore than 4 times the second surface area; the bed height is at least 10and not more than 200 millimetre; the bed volume is at least 10millilitre and not more than 20 litre; the diameter of the inner frit isat least 10 and below 150 millimetre; in the space delimited by theinner frit, a core member is located, the external wall of whichdelimits an inner flow channel with the inner frit; the outer flowchannel delimited outside the outer frit has a width at least 0.5millimetre and the inner flow channel has a width that equals the outerflow channel width times the actual I/O ratio.
 18. The method accordingto claim 16, wherein the inlet and outlet frit comprise exactly threewoven wire layers, mutually diffusion bonded, the filter layer directlyexposed to the torus shaped packed bed, the layer providing the oppositeface being a reinforcement layer, the layer in between being adispersion or reinforcement layer.
 19. The method according to claim 18,the inner and outer frit are made from exactly three layers of wovenwires of stainless steel, directly laid on top of each other, andproviding a pore size of at least 100 micrometre and the bed height isat least 20 and below 150 millimetre and the hydrophilic beads have asize between 200 micrometre and 1 millimetre and the I/O is below 3.5:1such that the first surface area is at least 1.5 and not more than 3.5times the second surface area.
 20. The method according to claim 19, thefilter layer of a frit is woven according to plain dutch weave. 21.Method of processing a process liquid using a set of mutually identicalliquid chromatography columns, each column is for liquid chromatographycomprising a torus shaped packed bed of beads, the process liquidcontaining biologics that are captured by the beads wherein as the firstprocessing step downstream from the process liquid creating source,being a cell culture vessel, the process liquid is fed to a subset of atleast two of the set of identical columns directly from the sourcewithout any intermediate filtration or other clarification equipment,nor any harvest holding-step or holding vessel and the process liquid,depleted by the subset of columns from the product of interest, isleaving said subset of columns after having flown radially through eachof the columns of the subset of columns, wherein the process liquid isconcentrated crude CHO cell culture harvest, the CHO cell diameter isapproximately 0.1-10% of bead diameter, the bead diameter isapproximately 120-500 micrometer, the beads are hydrophilic; each liquidchromatography column of the set is designed to be radially flownthrough by the process liquid and comprising the packed bed of beadsdesigned to capture product from the process liquid, the packed bed heldbetween an inlet and outlet frit of the column wherein the inlet fritthrough which the liquid to be processed enters the packed bed, has afirst surface area and wherein the outlet frit through which the liquidexits the packed bed has a second surface area smaller than the firstsurface area, wherein the column has an I/O ratio which is the ratiobetween the surface area of the inlet frit and the surface area of theoutlet frit, and wherein the liquid to be processed flows radiallyinward from the inlet frit towards the outlet frit; the columns of thesubset of at least two columns are connected in series with each otherand the process liquid is supplied to the most upstream column of thesubset, flows successively through the in series connected columns andleaves the subset through the most downstream column and after timeelapse, if the most upstream column is become saturated with product,the most upstream column is disconnected from the subset and removedfrom the series connection and a replacement column from the set isadded to the subset such that it is connected in series downstream fromthe most downstream column of the subset and this process is repeatedand the column removed from the subset is off-line processed to resetthe packed bed of this column such that this column is made ready tobecome available for the subset to become saturated again by connectingthis column in series as the most downstream column of the subset;wherein the most upstream column of the subset is connected directly tothe cell culture exit of the cell culture vessel in which cell culturingtakes place and which cell culture vessel contains between 1,000 and10,000 litre of mixture of cell culture media and the crude CHO cellculture harvest and wherein for each column the following applies: theI/O ratio is at least 1.5:1 and not more than 3.5:1, such that the firstsurface area is at least 1.5 and not more than 3.5 times the secondsurface area; the bed height is at least 10 and not more than 200millimetre; the bed volume is at least 10 millilitre and not more than20 litre; the diameter of the inner frit is at least 10 and below 150millimetre; in the space delimited by the inner frit, a core member islocated, the external wall of which delimits an inner flow channel withthe inner frit; the outer flow channel delimited radially outside theouter frit has a width at least 0.5 millimetre and the inner flowchannel has a width that equals the outer flow channel width times theactual I/O ratio.
 22. The method according to claim 21, wherein theinlet and outlet frit each comprise exactly three woven wire layers ofstainless steel, mutually diffusion bonded and directly laid on top opeach other, of which the filter layer provides and outer layer of thefrit and which filter layer is directly exposed to the torus shapedpacked bed, of which the layer providing the opposite outer face of thefrit being a reinforcement layer, and of which the layer in betweenbeing a dispersion layer and each frit providing a pore size of at least100 micrometre; and the bed height is at least 20 and below 150millimetre and the hydrophilic beads have a size between 200 micrometreand 1 millimetre and the filter layer of a frit is woven according toplain dutch weave.